The Exploration Of SD Rat And Rhesus Monkey Anaphlaxis About Huttuynia Cordata Injection

Reevaluate the safety of Huttuynia Cordata injection with SD rat. And in order to establish the methods of SYBR II real-time fluorescent quantitative RT-PCR for determining the expression levels of cytokine in macaque and apply for sensitivity response of macaques by these methods. Reevaluate the safety of Huttuynia Cordata injection with macaque. And with the results above all, explore the differentiation of SD rat and macaque in sensitivity test.SD rat was used as experimental animal to evaluate the sensitization of Huttaynia Cordata injection by active systemic allergic reaction. Determine the contents of HIS 、IgE and IgG by ELISA kits. Quantitative real-time PCR examination the mRNA expression level of IL-4、IL-10、IL-13 and IFN-γ. According to the sequences of IL-4,IL-10,IL-13,IFN-y and reference gene β-actin from GenBank, five pairs of specific primers were designed respectively. Total RNA was extracted with trizol from macaque PBMCs and mRNA was reverse-transcribed into cDNA. Then quantitative real-time PCR methods were established to evaluate the relative expression levels of the four cytokines, respectively. And macaque was used as experiment animal, to evaluate the sensitization of Huttuynia Cordata injection by active systemic allergic reaction. Determine the contents of HIS、IgE and IgG by ELISA kits. Quantitative real-time PCR examination the mRNA expression level of IL-4、L-10、IL-13 and IFN-y.The active systemic allergic reaction results showed that the positive control group was strongly positive in SD rat.The results of Huttuynia Cordata distillate was negative in SD rat. And the Huttuynia Cordata injection A group and the Huttuynia Cordata injection B group were both weakly positive in SD rat. To the Huttuynia Cordata injection A group and the Huttuynia Cordata injection B group, the increasing of IgE and HIS in SD rat was not significant(P>0.05). In the SD rat assays injected with Huttuynia Cordata distillate, the mRNA expression level of IL-4、IL-10、IL-13 and IFN-γ were not increased significantly (P>0.05), and injected with Huttuynia Cordata injection A and B, the levels in IL-4、IL-10、 IL-13 and IFN-γ were increased significantly (PO.01,P<0.05). the real-time PCR method was developed with high sensitivity (the detection dilution was 102 copies/μL) and good specificity and repeatability(the intra-assay CV of β-actin was below 0.02, and inter-assay CV was between 0.018 and 0.060; the intra-assay CV of IL-4 was below 0.04, and inter-assay CV was between 0.032 and 0.042; the intra-assay CV of IL-10 was below 0.04, and inter-assay CV was between 0.039 and 0.057; the intra-assay CV of IL-13 was below 0.02, and inter-assay CV was between 0.017 and 0.026; the intra-assay CV of IFN-ybelow 0.02, and inter-assay CV was between 0.018and 0.062)in detecting macaque cytokines. The melting curve displayed a single peak. The active systemic allergic reaction results showed that the positive control group was strongly positive in macaque. The results of Huttuynia Cordata distillate were negative in macaque. The Huttuynia Cordata injection A group was weakly positive in macaque, but the Huttuynia Cordata injection B group was positive in macaque. To the Huttuynia Cordata injection A group and the Huttuynia Cordata injection B group, the contents of IgE and HIS were increased significantly in macaque(P<0.01). In the macaque allergy assays injected with Huttuynia Cordata distillate, the mRNA expression level of IL-4、IL-10、IL-13 and IFN-γ were increased significantly(P>0.01), and injected with Huttuynia Cordata injection A and B, the levels in IL-4、IL-10 and IL-13 were increased (P>0.05), but IFN-γ were decreased (P>0.05)Huttuynia Cordata distillate will not cause allergic reaction, but Huttuynia Cordata injection will. And macaque will be a better choice in the active systemic allergic reaction experiment. Second, these real time PCR methods could be applied for measuring the mRNA expression level of cytokines IL-4, IL-10, IL-13 and IFN-γ.